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The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid β aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies., The calcium binding protein S100B is an abundantly expressed protein in the brain and has neuro-protective functions by inhibiting Aβ aggregation and metal ion toxicity. Here, the authors combine cell biology and biochemical experiments with chemical kinetics and NMR measurements and show that S100B protein is an extracellular Tau chaperone and further characterize the interactions between S100B and Tau.

International audience; The protein-protein interactions (PPIs) of 14-3-3 proteins are a model system for studying PPI stabilization. The complex natural product Fusicoccin A stabilizes many 14-3-3 PPIs but is not amenable for use in SAR studies, motivating the search for more drug-like chemical matter. However, drug-like 14-3-3 PPI stabilizers enabling such study have remained elusive. An X-ray crystal structure of a PPI in complex with an extremely low potency stabilizer uncovered an unexpected non-protein interacting, ligand-chelated Mg 2+ leading to the discovery of metal ion-dependent 14-3-3 PPI stabilization potency. This originates from a novel chelation-controlled bioactive conformation stabilization effect. Metal chelation has been associated with pan-assay interference compounds (PAINS) and frequent hitter behavior, but chelation can evidently also lead to true potency gains and find use as a medicinal chemistry strategy to guide compound optimization. To demonstrate this, we exploited the effect to design the first potent, selective and drug-like 14-3-3 PPI stabilizers.

Protein-protein interactions (PPIs) remain poorly explored targets for the treatment of Alzheimer’s disease (AD). The interaction of 14-3-3 proteins with Tau was shown to have detrimental effects on neuronal cells and to be linked to Tau pathology. This PPI is therefore seen as a potential target for AD. When Tau is phosphorylated by PKA (Tau-PKA), two 14-3-3 binding epitopes are generated, surrounding the phosphorylated serines 214 and 324 of Tau. The crystal structures of 14-3-3 in complex with peptides surrounding these Tau phosphosites show that both these motifs are anchored in the amphipathic binding groove of 14-3-3. However, in the absence of structural data with the full-length Tau protein, the stoichiometry of the complex or the interface and affinity of the partners, are still unclear. In this work, we addressed these points, using a broad range of biophysical techniques. The interaction of the long disordered Tau-PKA protein with 14-3-3σ is restricted to two short sequences, containing phosphorylated serines, which bind in the amphipathic binding groove of 14-3-3. Phosphorylation of Tau is fundamental for the formation of this stable complex, and the affinity of the Tau-PKA/14-3-3 interaction is in the 1-10 micromolar range. Each monomer of the 14-3-3σ dimer binds one of the two different phosphorylated peptides of Tau-PKA, suggesting a 14-3-3/Tau-PKA stoichiometry of 2:1, confirmed by analytical ultracentrifugation. These results contribute to a better understanding of this PPI and provide useful insights for drug discovery projects aiming at the inhibition of this interaction.

International audience; Natural compounds are an important class of potent drug molecules including some retrospectively found to act as stabilizers of protein–protein interactions (PPIs). However, the design of synthetic PPI stabilizers remains an understudied approach. To date, there are limited examples where cooperativity has been utilized to guide the optimization of a PPI stabilizer. The 14-3-3 scaffold proteins provide an excellent platform to explore PPI stabilization because these proteins mediate several hundred PPIs, and a class of natural compounds, the fusicoccanes, are known to stabilize a subset of 14-3-3 protein interactions. 14-3-3 has been reported to negatively regulate the p65 subunit of the NF-κB transcription factor, which qualifies this protein complex as a potential target for drug discovery to control cell proliferation. Here, we report the high-resolution crystal structures of two 14-3-3 binding motifs of p65 in complex with 14-3-3. A semisynthetic natural product derivative, DP-005, binds to an interface pocket of the p65/14-3-3 complex and concomitantly stabilizes it. Cooperativity analyses of this interaction, and other disease relevant 14-3-3-PPIs, demonstrated selectivity of DP-005 for the p65/14-3-3 complex. The adaptation of a cooperative binding model provided a general approach to characterize stabilization and to assay for selectivity of PPI stabilizers.

International audience; Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein–protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as “undruggable” targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products.

Lamin B Receptor (LBR) is an integral protein of the interphase inner nuclear membrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has been extensively studied, very little is known about other post-translational modifications of the protein. There is only one report on the O-β-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites by using as substrates of O-β-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA binding while it marginally affects RS domain phosphorylation mediated by SRPK1, Akt2 and cdk1 kinases. General significance Our methodology providing a quantitative description of O-GlcNAc patterns based on a combination of mass spectrometry and high resolution NMR spectroscopy on short peptide substrates allows subsequent functional analyses. Hence, our approach is of general interest to a wide audience of biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation.

Metal ions are well known modulators of protein aggregation and are key players in Alzheimer&rsquo, s Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.

Insights into tau molecular structures have advanced significantly in recent years. This field has been the subject of recent breakthroughs, including the first cryo-electron microscopy structures of tau filaments from Alzheimer's and Pick's disease inclusions, as well as the structure of the repeat regions of tau bound to microtubules. Tau structure covers various species as the tau protein itself takes many forms. We will here address a range of studies that help to define the many facets of tau protein structures and how they translate into pathogenic forms. New results shed light on previous data that need now to be revisited in order to up-date our knowledge of tau molecular structure. Finally, we explore how these data can contribute the important medical aspects of this research - diagnosis and therapeutics. ispartof: ACTA NEUROPATHOLOGICA COMMUNICATIONS vol:7 issue:1 ispartof: location:England status: published

Although Tau is an intrinsically disordered protein, some level of structure can still be defined, corresponding to short stretches of dynamic secondary structures and a preferential global fold described as an ensemble of conformations. These structures can be modified by Tau phosphorylation, and potentially other post-translational modifications. The analytical capacity of Nuclear Magnetic Resonance (NMR) spectroscopy provides the advantage of offering a residue-specific view of these modifications, allowing to link specific sites to a particular structure. The cis or trans conformation of X-Proline peptide bonds is an additional characteristic parameter of Tau structure that is targeted and modified by prolyl cis/trans isomerases. The challenge in molecular characterization of Tau lies in being able to link structural parameters to functional consequences in normal functions and dysfunctions of Tau, including potential misfolding on the path to aggregation and/or perturbation of the interactions of Tau with its many molecular partners. Phosphorylation of Ser and Thr residues has the potential to impact the local and global structure of Tau.

Current molecular hypotheses have not yet delivered marketable treatments for Alzheimer's disease (AD), arguably due to a lack of understanding of AD biology and an overreliance on conventional drug modalities. Protein-protein interactions (PPIs) are emerging drug targets, which show promise for the treatment of, e.g., cancer, but are still underexploited for treating neurodegenerative diseases. 14-3-3 binding to phosphorylated Tau is a promising PPI drug target based on its reported destabilizing effect on microtubules, leading to enhanced neurofibrillary tangle formation as a potential cause of AD-related neurodegeneration. Inhibition of 14-3-3/Tau may therefore be neuroprotective. Previously, we reported the structure-guided development of modified peptide inhibitors of 14-3-3/Tau. Here, we report further efforts to optimize the binding mode and activity of our modified Tau peptides through a combination of chemical synthesis, biochemical assays, and X-ray crystallography. Most notably, we were able to characterize two different high-affinity binding modes, both of which inhibited 14-3-3-binding to full-length PKA-phosphorylated Tau protein in vitro as measured by NMR spectroscopy. Our findings, besides producing useful tool inhibitor compounds for studying 14-3-3/Tau, have enhanced our understanding of the molecular parameters for inhibiting 14-3-3/Tau, which are important milestones toward the establishment of our 14-3-3 PPI hypothesis.

International audience; MED26 is a subunit of Mediator, a large complex central to the regulation of gene transcription by RNA Polymerase II. MED26 plays a role in the switch between the initiation and elongation phases of RNA Polymerase II-mediated transcription process. Regulation of these steps requires successive binding of MED26 N-terminal domain (NTD) to TATA-binding protein-associated factor 7 (TAF7) and Eleven-nineteen lysine-rich in leukemia-Associated Factor 1 (EAF1). In order to investigate the mechanism of regulation by MED26, MED26-NTD structure was solved by NMR, revealing a 4-helix bundle. EAF1 (239-268) and TAF7 (205-235) peptide interactions were both mapped to the same groove formed by H3 and H4 helices of MED26-NTD. Both interactions are characterized by dissociation constants in the 10-μM range. Further experiments revealed a folding-upon-binding mechanism that leads to the formation of EAF1 (N247-S260) and TAF7 (L214-S227) helices. Chemical shift perturbations and nuclear Overhauser enhancement contacts support the involvement of residues I222/F223 in anchoring TAF7 helix to a hydrophobic pocket of MED26-NTD, including residues L48, W80 and I84. In addition, Ala mutations of charged residues located in the C-terminal disordered part of TAF7 and EAF1 peptides affected the binding, with a loss of affinity characterized by a 10-time increase of dissociation constants. A structural model of MED26-NTD/TAF7 complex shows bi-partite components, combining ordered and disordered segments, as well as hydrophobic and electrostatic contributions to the binding. This study provides molecular detail that will help to decipher the mechanistic basis for the initiation to elongation switch-function mediated by MED26-NTD.

International audience; Bridging integrator 1 (bin1) gene is a genetic determinant of Alzheimer's disease (AD) and has been reported to modulate Alzheimer's pathogenesis through pathway(s) involving Tau. The functional impact of Tau/BIN1 interaction as well as the molecular details of this interaction are still not fully resolved. As a consequence, how BIN1 through its interaction with Tau affects AD risk is also still not determined. To progress in this understanding, interaction of Tau with two BIN1 isoforms was investigated using Nuclear Magnetic Resonance spectroscopy. 1H, 15N spectra showed that the C-terminal SH3 domain of BIN1 isoform 1 (BIN1Iso1) is not mobile in solution but locked with the core of the protein. In contrast, the SH3 domain of BIN1 isoform 9 (BIN1Iso9) behaves as an independent mobile domain. This reveals an equilibrium between close and open conformations for the SH3 domain. Interestingly, a 334–376 peptide from the clathrin and AP-2-binding domain (CLAP) domain of BIN1Iso1, which contains a SH3-binding site, is able to compete with BIN1-SH3 intramolecular interaction. For both BIN1 isoforms, the SH3 domain can interact with Tau(210–240) sequence. Tau(210–240) peptide can indeed displace the intramolecular interaction of the BIN1-SH3 of BIN1Iso1 and form a complex with the released domain. The measured Kd were in agreement with a stronger affinity of Tau peptide. Both CLAP and Tau peptides occupied the same surface on the BIN1-SH3 domain, showing that their interaction is mutually exclusive. These results emphasize an additional level of complexity in the regulation of the interaction between BIN1 and Tau dependent of the BIN1 isoforms.

International audience; Determining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205.

International audience; We study the aggregation of a fragment of the neuronal protein Tau that contains part of the proline rich domain and of the microtubule binding repeats. When incubated at 37 °C with heparin, the fragment readily forms fibers as witnessed by Thioflavin T fluorescence. Electron microscopy and NMR spectroscopy show bundled ribbon like structures with most residues rigidly incorporated in the fibril. Without its cysteines, this fragment still forms fibers of a similar morphology, but with lesser Thioflavin T binding sites and more mobility for the C-terminal residues.

International audience; Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis of in vitro phosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites. In vitro phosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors., The response to hypoxia involves multiple genes regulated by the hypoxia inducible transcription factors (HIFs), whose stability is regulated by prolyl hydroxylation. Here the authors provide a molecular basis for the substrate selectivity of the HIF prolyl hydroxylases that can be altered in erythrocytosis and cancer.

International audience; The aggregation of the neuronal Tau protein is one molecular hallmark of Alzheimer's disease and other related tauopathies, but the precise molecular mechanisms of the aggregation process remain unclear. The FK506 binding protein FKBP52 is able to induce oligomers in the pathogenic Tau P301L mutant and in a truncated form of the wild-type human Tau protein. Here, we investigate whether FKBP52's capacity to induce Tau oligomers depends on its prolyl cis/trans isomerase activity. We find that FKBP52 indeed can isomerize selected prolyl bonds in the different Tau proteins, and that this activity is carried solely by its first FK506 binding domain. Its capacity to oligomerize Tau is however not linked to this PPIase activity. In addition, we identified a novel molecular interaction implying the PHF6 peptide of Tau and the FK1/FK2 domains of FKBP52 independent of FK506 binding; these data point towards a non-catalytic molecular interaction that might govern the effect of FKBP52 on Tau.

The conformational state of distinct prolines can determine the folding of a protein but equally other biological processes when coupled to a conformation-sensitive secondary reaction. For the neuronal tau protein, the importance of proline conformation is underscored by its interaction with different prolyl cis/trans isomerases. The proline conformation would gain even further importance after phosphorylation of the preceding residue by various proline-directed kinases. A number of molecular diseases including Alzheimer's disease and traumatic brain injury were thereby recently qualified as "cistauosis", as they would imply a cis conformation for the pThr231-Pro232 prolyl bond. We here investigate by NMR spectroscopy the conformation of all prolines in a functional Tau fragment, Tau[208-324]. Although we can detect and identify some minor conformers in the cis form, we show that all prolines are for over 90% in the trans conformation. Phosphorylation by CDK2/CycA3, which notably leads to complete modification of the Thr231 residue, does not change this conclusion. Our data hence disagree with the notion that specific prolyl bonds in tau would adopt preferentially the cis conformation.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed. 118

International audience; We report a biochemical and structural characterization of domain 2 of the non-structural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with Cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to Cyclosporin A (CsA) resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both Cyclophilins. The interaction surface on the Cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.

14-3-3 proteins act as adapters that exert their function by interacting with their various protein partners. 14-3-3 proteins have been implicated in a variety of human diseases including neurodegenerative diseases. 14-3-3 proteins have recently been reported to be abundant in the neurofibrillary tangles (NFTs) observed inside the neurons of brains affected by Alzheimer's disease (AD). These NFTs are mainly constituted of phosphorylated Tau protein, a microtubule-associated protein known to bind 14-3-3. Despite this indication of 14-3-3 protein involvement in the AD pathogenesis, the role of 14-3-3 in the Tauopathy remains to be clarified. In the present study, we shed light on the role of 14-3-3 proteins in the molecular pathways leading to Tauopathies. Overexpression of the 14-3-3s isoform resulted in a disruption of the tubulin cytoskeleton and prevented neuritic outgrowth in neurons. NMR studies validated the phosphorylated residues pSer214 and pSer324 in Tau as the 2 primary sites for 14-3-3 binding, with the crystal structure of 14-3-3s in complex with Tau-pSer214 and Tau-pSer324 revealing the molecular details of the interaction. These data suggest a rationale for a possible pharmacologic intervention of the Tau/14-3-3 interaction.-Joo, Y., Schumacher, B., Landrieu, I., Bartel, M., Smet-Nocca, C., Jang, A., Choi, H. S., Jeon, N. L., Chang, K.-A., Kim, H.-S., Ottmann, C., Suh, Y.-H. Involvement of 14-3-3 in tubulin instability and impaired axon development is mediated by Tau.

Introduction The application of high-throughput genomic approaches has revealed 24 novel risk loci for Alzheimer’s disease (AD). We recently reported that the bridging integrator 1 (BIN1) risk gene is linked to Tau pathology. Results We used glutathione S-transferase pull-down assays and nuclear magnetic resonance (NMR) experiments to demonstrate that BIN1 and Tau proteins interact directly and then map the interaction between BIN1’s SH3 domain and Tau’s proline-rich domain (PRD) . Our NMR data showed that Tau phosphorylation at Thr231 weakens the SH3-PRD interaction. Using primary neurons, we found that BIN1-Tau complexes partly co-localize with the actin cytoskeleton; however, these complexes were not observed with Thr231-phosphorylated Tau species. Conclusion Our results show that (i) BIN1 and Tau bind through an SH3-PRD interaction and (ii) the interaction is downregulated by phosphorylation of Tau Thr231 (and potentially other residues). Our study sheds new light on regulation of the BIN1/Tau interaction and opens up new avenues for exploring its complex’s role in the pathogenesis of AD. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0237-8) contains supplementary material, which is available to authorized users.

© 2015 Elsevier Ltd. All rights reserved. Cyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme that plays numerous roles, notably in brain development. CDK5 is activated through its association with the activators, p35 and p39, rather than by cyclins. Proteolytic procession of the N-terminal part of its activators has been linked to Alzheimer's disease and various other neuropathies. The interaction with the proteolytic product p25 prolongs CDK5 activation and modifies the substrate specificity. In order to discover small-molecule inhibitors of the interaction between CDK5 and p25, we have used a bioluminescence resonance energy transfer (BRET)-based screening assay. Among the 1,760 compounds screened, the generic drug tamoxifen has been identified. The inhibition of the CDK5 activity by tamoxifen was notably validated by monitoring the phosphorylation state of tau protein. The study of the molecular mechanism of inhibition indicates that tamoxifen interacts with p25 to block the CDK5/p25 interaction and pave the way for new treatments of tauopathies.

Tauopathies, including Alzheimer's disease (AD), are neurodegenerative diseases associated with the pathologic aggregation of human brain Tau protein. Neuronal Tau is involved in microtubule (MT) formation and stabilization. We showed previously that the immunophilin FK506-binding protein of MW ∼52 kDa (FKBP52) interferes with this function of full-length Tau and provokes aggregation of a disease-related mutant of Tau. To dissect the molecular interaction between recombinant human FKBP52 and Tau, here, we study the effect of FKBP52 on a functional Tau fragment (Tau-F4, Ser(208)-Ser(324)) containing part of the proline- rich region and MT-binding repeats. Therefore, we perform MT assembly and light-scattering assays, blue native PAGE analysis, electron microscopy, and Tau seeding experiments in SH-SY5Y human neuroblastoma cells. We show that FKBP52 (6 µM) prevents MT formation generated by Tau-F4 (5 µM) and induces Tau-F4 oligomerization and aggregation. Electron microscopy analyses show granular oligomers and filaments of Tau-F4 after short-time FKBP52 incubation. We demonstrate that the terminal parts of Tau interfere with the effects of FKBP52. Finally, we find that FKBP52-induced Tau-F4 oligomers cannot only generate in vitro, direct conformational changes in full-length Tau and that their uptake into neuronal cells can equally lead to aggregation of wild-type endogenous Tau. This suggests a potential prion-like property of these particular Tau-F4 aggregates. Collectively, our results strengthen the hypothesis of FKBP52 involvement in the Tau pathogenicity process.

Post mortem biochemical staging of Alzheimer’s disease is currently based on immunochemical analysis of brain slices with the AT8 antibody. The epitope of AT8 is described around the pSer202/pThr205 region of the hyperphosphorylated form of the neuronal protein tau. In this study, NMR spectroscopy was used to precisely map the AT8 epitope on phosphorylated tau, and derive its defining structural features by a combination of NMR analyses and molecular dynamics. A particular turn conformation is stabilized by a hydrogen bond of the phosphorylated Thr205 residue to the amide proton of Gly207, and is further stabilized by the two Arg residues opposing the pSer202/pThr205. 54;23

The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions. The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions. The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions.

Interactions involving phosphorylated Ser/Thr-Pro motifs in proteins play a key role in numerous regulatory processes in the cell. Here, we investigate potential ligands of the WW binding domain of Pin1 in order to inhibit protein-protein interactions between Pin1 and phosphopeptides. Our structure-based strategy implies the synthesis of analogues of the Ac-Thr(PO(3)H(2))-Pro-NH(2) dipeptide and relies on high resolution NMR spectroscopy to accurately measure the affinity constants even in the high micromolar range.

The regulation of Plasmodium falciparum protein phosphatase type 1 (PfPP1) activity remains to be deciphered. Data from homologous eukaryotic type 1 protein phosphatases (PP1) suggest that several protein regulators should be involved in this essential process. One such regulator, named PfI2 based on its primary sequence homology with eukaryotic inhibitor 2 (I2), was recently shown to be able to interact with PfPP1 and to inhibit its phosphatase activity, mainly through the canonical ‘RVxF’ binding motif. The details of the structural and functional characteristics of this interaction are investigated here. Using NMR spectroscopy, a second site of interaction is suggested to reside between residues D94 and T117 and contains the ‘FxxR/KxR/K’ binding motif present in other I2 proteins. This site seems to play in concert/synergy with the ‘RVxF’ motif to bind PP1, because only mutations in both motifs were able to abolish this interaction completely. However, regarding the structure/function relationship, mutation of either the ‘RVxF’ or ‘FxxR/KxR/K’ motif is more drastic, because each mutation prevents the capacity of PfI2 to trigger germinal vesicle breakdown in microinjected Xenopus oocytes. This indicates that the tight association of the PfI2 regulator to PP1, mediated by a two-site interaction, is necessary to exert its function. Based on these results, the use of a peptide derived from the ‘FxxR/KxR/K’ PfI2 motif was investigated for its potential effect on Plasmodium growth. This peptide, fused at its N-terminus to a penetrating sequence, was shown to accumulate specifically in infected erythrocytes and to have an antiplasmodial effect. Structured digital abstract PfPP1 and PfI2 bind by nuclear magnetic resonance (1, 2) PP1 physically interacts with PfPP1 by anti tag coimmunoprecipitation (View interaction) PfI2 binds to PfPP1 by enzyme linked immunosorbent assay (View interaction) PfI2 binds to PfPP1 by surface plasmon resonance (1, 2, 3, 4)

International audience; Lysine acetylation of the neuronal Tau protein was described as a novel mechanism of posttranslational regulation of Tau functions with important outcomes in microtubule binding and aggregation processes related to Alzheimer's disease. Here, we unravel at a per-residue resolution the acetylation pattern of full-length Tau by the Creb-binding protein (CBP) acetyltransferase using high-resolution nuclear magnetic resonance spectroscopy. Our study gives a quantitative overview of CBP-mediated acetylation and examines the catalytic proficiency because the nonenzymatic reaction with acetyl-coenzyme A occurs in vitro. Furthermore, we have investigated with this characterized acetylated Tau the effect of acetylation on Tau fibrillization in a heparin-induced aggregation assay and on heparin binding.

International audience; The Tau protein is the major component of intracellular filaments observed in a number of neurodegenerative diseases known as tauopathies. The pathological mutant of Tau containing a proline-to-leucine mutation at position 301 (P301L) leads to severe human tauopathy. Here, we assess the impact of FK506-binding protein with a molecular mass of ∼52 kDa (FKBP52), an immunophilin protein that interacts with physiological Tau, on Tau-P301L activity. We identify a direct interaction of FKBP52 with Tau-P301L and its phosphorylated forms and demonstrate FKBP52's ability to induce the formation of Tau-P301L oligomers. EM analysis shows that Tau-P301L oligomers, induced by FKBP52, can assemble into filaments. In the transgenic zebrafish expressing the human Tau-P301L mutant, FKBP52 knockdown is sufficient to redrive defective axonal outgrowth and branching related to Tau-P301L expression in spinal primary motoneurons. This result correlates with a significant reduction of pT181 pathological phosphorylated Tau and with recovery of the stereotypic escape response behavior. Collectively, FKBP52 appears to be an endogenous candidate that directly interacts with the pathogenic Tau-P301L and modulates its function in vitro and in vivo.

A homologue of the human site-specific prolyl cis/trans isomerase PIN1 was identified in Arabidopsis thaliana. The PIN1At gene encodes a protein of 119 amino acids that is 53% identical with the catalytic domain of the human PIN1 parvulin. Steady-state PIN1At mRNA is found in all plant tissues tested. We show by two-dimensional NMR spectroscopy that the PIN1At is a prolyl cis/trans isomerase with specificity for phosphoserine-proline bonds. PIN1At is the first example of an eukaryotic parvulin without N- or C-terminal extensions. The N-terminal WW domain of 40 amino acids, typical of all the phosphorylation-dependent eukaryotic parvulins, is absent. However, triple-resonance NMR experiments showed that PIN1At contained a hydrophobic helix similar to the alpha1 helix observed in PIN1 that could mediate the protein-protein interactions.

International audience; A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.

International audience; We describe our efforts to combine in vitro enzymatic reactions with recombinant kinases to phosphorylate the neuronal tau protein, and NMR spectroscopy to unravel the resulting phosphorylation pattern in both qualitative and quantitative manners. This approach, followed by functional assays with the same samples, gives access to the complex phosphorylation code of tau. As a result, we propose a novel hypothesis for the link between tau (hyper)phosphorylation and aggregation.

16 páginas, 9 figuras. Contiene en material suplementario: Figs. S1–S3 and Tables S1 and S2."This research was originally published in Journal of Biological Chemistry. Fréville A, Landrieu I, García-Gimeno MA, Vicogne J, Montbarbon M, Bertin B, Verger A, Kalamou H, Sanz P, Werkmeister E, Pierrot C, Khalife J. Plasmodium falciparum inhibitor-3 homolog increases protein phosphatase type 1 activity and is essential for parasitic survival. J Biol Chem. 2012 Jan 6;287(2):1306-21. © the American Society for Biochemistry and Molecular Biology", Growing evidence indicates that the protein regulators governing protein phosphatase 1 (PP1) activity have crucial functions because their deletion drastically affects cell growth and division. PP1 has been found to be essential in Plasmodium falciparum, but little is known about its regulators. In this study, we have identified a homolog of Inhibitor-3 of PP1, named PfI3. NMR analysis shows that PfI3 belongs to the disordered protein family. High affinity interaction of PfI3 and PfPP1 is demonstrated in vitro using several methods, with an apparent dissociation constant K(D) of 100 nm. We further show that the conserved (41)KVVRW(45) motif is crucial for this interaction as the replacement of the Trp(45) by an Ala(45) severely decreases the binding to PfPP1. Surprisingly, PfI3 was unable to rescue a yeast strain deficient in I3 (Ypi1). This lack of functional orthology was supported as functional assays in vitro have revealed that PfI3, unlike yeast I3 and human I3, increases PfPP1 activity. Reverse genetic approaches suggest an essential role of PfI3 in the growth and/or survival of blood stage parasites because attempts to obtain knock-out parasites were unsuccessful, although the locus of PfI3 is accessible. The main localization of a GFP-tagged PfI3 in the nucleus of all blood stage parasites is compatible with a regulatory role of PfI3 on the activity of nuclear PfPP1., The NMR facilities were funded by the Région Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry, and the University of Sciences and Technologies of Lille I.This work was supported in part by Inserm, Institut Pasteur de Lille, Université Lille Nord de France, and TGE RMN THC Grant FR-3050, France

International audience; We present here the characterization of the epitope recognized by the AT180 monoclonal antibody currently used to define an Alzheimer's disease (AD)-related pathological form of the phosphorylated Tau protein. Some ambiguity remains as to the exact phospho-residue(s) recognized by this monoclonal: pThr231 or both pThr231 and pSer235. To answer this question, we have used a combination of nuclear magnetic resonance (NMR) and fluorescence spectroscopy to characterize in a qualitative and quantitative manner the phospho-residue(s) essential for the epitope recognition. Data from the first step of NMR experiments are used to map the residues bound by the antibodies, which were found to be limited to a few residues. A fluorophore is then chemically attached to a cystein residue introduced close-by the mapped epitope, at arginine 221, by mutagenesis of the recombinant protein. The second step of Förster resonance energy transfer (FRET) between the AT180 antibody tryptophanes and the phospho-Tau protein fluorophore allows to calculate a dissociation constant Kd of 30nm. We show that the sole pThr231 is necessary for the AT180 recognition of phospho-Tau and that phosphorylation of Ser235 does not interfere with the binding.

International audience; Pin1 is a prolyl isomerase that recognizes phosphorylated Ser/Thr-Pro sites, and phosphatase inhibitor-2 (I-2) is phosphorylated during mitosis at a PSpTP site that is expected to be a Pin1 substrate. However, we previously discovered I-2, but not phospho-I-2, bound to Pin1 as an allosteric modifier of Pin1 substrate specificity [Li, M., et al. (2008) Biochemistry 47, 292]. Here, we use binding assays and NMR spectroscopy to map the interactions on Pin1 and I-2 to elucidate the organization of this complex. Despite having sequences that are ∼50% identical, human, Xenopus, and Drosophila I-2 proteins all exhibited identical, saturable binding to GST-Pin1 with K(0.5) values of 0.3 μM. The (1)H-(15)N heteronuclear single-quantum coherence spectra for both the WW domain and isomerase domain of Pin1 showed distinctive shifts upon addition of I-2. Conversely, as shown by NMR spectroscopy, specific regions of I-2 were affected by addition of Pin1. A single-residue I68A substitution in I-2 weakened binding to Pin1 by half and essentially eliminated binding to the isolated WW domain. On the other hand, truncation of I-2 to residue 152 had a minimal effect on binding to the WW domain but eliminated binding to the isomerase domain. Size exclusion chromatography revealed that wild-type I-2 and Pin1 formed a large (>300 kDa) complex and I-2(I68A) formed a complex of half the size that we propose are a heterotetramer and a heterodimer, respectively. Pin1 and I-2 are conserved among eukaryotes from yeast to humans, and we propose they make up an ancient partnership that provides a means for regulating Pin1 specificity and function.

International audience; Nonstructural protein 5A (NS5A) is essential for Hepatitis C Virus (HCV) replication and constitutes an attractive target for antiviral drug development. While structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit propensity to partially fold into an alpha-helix. NMR analysis identifies two putative alpha-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element (MoRE), and as such promote the interaction with relevant biological partner(s). As mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and Cyclophilin A (CypA). CypA indeed interacts with NS5A-D3 and this interaction is completely abolished by Cyclosporin A (CsA). NMR heteronuclear exchange experiments demonstrate that CypA has in vitro PPIase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.

International audience; Phosphorylation of the microtubule-associated Tau protein plays a major role in the regulation of its activity of tubulin polymerization and/or stabilization of microtubule assembly. A dysregulation of the phosphorylation/dephosphorylation balance leading to the hyperphosphorylation of Tau proteins in neurons is thought to favor their aggregation into insoluble filaments. This in turn might underlie neuronal death as encountered in many neurodegenerative disorders, including Alzheimer's disease. Another post-translational modification, the O-linked β-N-acetylglucosaminylation (O-GlcNAcylation), controls the phosphorylation state of Tau, although the precise mechanism is not known. Moreover, analytical difficulties have hampered the precise localization of the O-GlcNAc sites on Tau, except for the S400 site that was very recently identified on the basis of ETD-FT-MS. Here, we identify three O-GlcNAc sites by screening a library of small peptides sampling the proline-rich, the microtubule-associated repeats and the carboxy-terminal domains of Tau as potential substrates for the O-β-N-acetylglucosaminyltransferase (OGT). The in vitro activity of the nucleocytoplasmic OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Using phosphorylated peptides, we establish the relationship between phosphate and O-GlcNAc incorporation at these sites. Phosphorylation of neighboring residues S396 and S404 was found to decrease significantly S400 O-GlcNAcylation. Reciprocally, S400 O-GlcNAcylation reduces S404 phosphorylation by the CDK2/cyclinA3 kinase and interrupts the GSK3β-mediated sequential phosphorylation process.

International audience; Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.

International audience; NMR spectroscopy was used to explore the different aspects of the normal and pathological functions of tau, but proved challenging because the protein contains 441 amino acids and has poor signal dispersion. We have set out to dissect the phosphorylation patterns of tau in order to understand better its role in the aggregation process and microtubule-binding regulation. Our current knowledge on the functional consequences of specific phosphorylations is still limited, mainly because producing and assessing quantitatively phosphorylated tau samples is far from straightforward, even in vitro. We use NMR spectroscopy as a proteomics tool to characterize the phosphorylation patterns of tau, after in vitro phosphorylation by recombinant kinases. The phosphorylated tau can next be use for functional assays or interaction assays with phospho-dependent protein partners, such as the prolyl cis-trans isomerase Pin1.

International audience; Debio 025 is a cyclosporin A (CsA) analogue that interferes strongly with the hepatitis C viral life cycle. Compared to CsA, Debio 025 has an additional methyl group at position 3 of the cyclic undecapeptide and an N-ethylvaline instead of an N-methylleucine at position 4. Unlike CsA, Debio 025 lacks immunosuppressive activity in vitro and in vivo. We show here that, in vitro, the cyclophilin A (CypA)-Debio 025 complex cannot interact any longer with calcineurin (CaN), a determinant for the immunosuppressive activity of CsA. We further use NMR spectroscopy to investigate at the molecular level the interaction of Debio 025 with CypA and thereby understand the basis for this loss of CaN interaction. NMR data and molecular modeling indicate that Debio 025 optimally interacts with CypA, which underlies the anti-HCV properties of Debio 025. However, the interaction between CaN and the CypA-Debio 025 complex is impeded by sterical hindrance of the CaN with the side chain of its Val4 residue. This is in sharp contrast with the case for the CypA-CsA-CaN ternary complex, where the Leu4 side chain can enter a hydrophobic cavity at the CaN interface. The structure of the CypA-Debio 025 complex thus provides a rational explanation for the non-immunosuppressive character of Debio 025.

Talin establishes a major link between integrins and actin filaments, and contains two distinct integrin binding sites, one, IBS1, located in the talin head domain and involved in integrin activation, and a second, IBS2, that we have mapped to helix 50 of the talin rod domain and that is essential for linking integrin beta subunits to the cytoskeleton (1 Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J Biol Chem 282, 17280-17288). Through the combined approach of mutational analysis of the beta3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies as well as site-specific antibody inhibition experiments, we provide evidence that the integrin beta3 - talin rod interaction relies on a helix - helix association between alpha-helix 50 of the talin rod domain and the membrane proximal alpha-helix of the beta3 integrin cytoplasmic tail, and that charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin beta3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the beta3 subunit cytoplasmic helix as the integrin alphaIIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the beta3 subunit following integrin activation.

The chemotaxis and integrin-mediated adhesion of T lymphocytes triggered by secreted cyclophilin B (CypB) depend on interactions with both cell surface heparan sulphate proteoglycans (HSPG) and the extracellular domain of the CD147 membrane receptor. Here, we use NMR spectroscopy to characterize the interaction of CypB with heparin-derived oligosaccharides. Chemical shift perturbation experiments allowed the precise definition of the heparan sulphate (HS) binding site of CypB. The N-terminal extremity of CypB, which contains a consensus sequence for heparin-binding proteins was modelled on the basis of our experimental NMR data. Because the HS binding site extends towards the CypB catalytic pocket, we measured its PPIase activity in the absence or presence of a HS oligosaccharide toward a CD147-derived peptide. We report the first direct evidence that CypB is enzymatically active on CD147, as it is able to accelerate the cis-trans isomerisation of the Asp179-Pro180 bond in a CD147-derived peptide. However, HS binding has no significant influence on this PPIase activity. We thus conclude that the glycanic moiety of HSPG serves as anchor for CypB at the cell surface, and that the signal could be transduced by CypB via its peptidyl-prolyl cis-trans isomerase (PPIase) activity toward CD147.

The crucial role of the neuronal Tau protein in microtubule stabilization and axonal transport suggests that too little or too much Tau might lead to neuronal dysfunction. The presence of a hyper phosphorylated but non aggregated molecule as a toxic species that might sequester normal Tau is discussed. We present recent in vitro results that might allow us to dissect the role of individual phosphorylation sites on its structure and function. We also discuss in this review the role of phosphorylation for the aggregation of the neuronal Tau protein, and compare it to the aggregation induced by external poly anions.

Background: Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). Like isoniazid, and pyrazinamide, ethionamide is a prodrug that needs to be activated by a mycobacterial enzyme. Activation pathways of prodrugs are generally problematic to uncover as they produce intermediates potentially difficult to characterize, to purify and that might prove unstable outside of their cellular context. Objectives and methods: We have used high resolution magic angle spinning-NMR (HRMAS-NMR) to follow ethionamide activation directly within living mycobacterial cells. Results: Data indicated that the intracellular metabolization of ethionamide strictly depends on the presence of the monooxygenase EthA and that EthA-dependent activation of ethionamide is coupled to a precise molecular sorting mechanism of the ethionamide metabolites. We found that the previously identified ethionamide metabolite 2-ethyl-4-hydroxymethylpyridine is produced in substantial amounts by the ethionamide-treated mycobacteria and that it is present exclusively outside of the bacteria. In contrast, the still unidentified ethionamide metabolite ETH* is the only ethionamide derivative detected within the bacterial cell. Moreover, ETH* appears to be unable to cross the bacterial envelope and consequently accumulates within the cytoplasm of the ethionamide-treated mycobacteria. Conclusions: These results strongly suggest that ETH* is the active antimycobacterial ethionamide derivative and open new perspectives for the understanding of the mode of action of prodrugs.

The neuronal Tau protein, whose physiological role is to stabilize the microtubules, is found under the form of aggregated filaments and tangles in Alzheimer's diseased neurons. Until recently detailed structural analysis of the natively unfolded Tau protein has been hindered due to its shear size and unfavourable amino acid composition. We review here the recent progress in the assignments of the full-length polypeptide using novel methods of product planes and peptide NMR mapping, and indicate the structural insights that can be obtained from this assignment. Preliminary NMR data on the fibers show that the assignment enables a precise mapping of the rigid core. Future NMR experiments should allow one to gain more insight into the conformational aspects of this intriguing protein.

PIN1 participates in the regulation of a number of signalling pathways in the cell involving protein phosphorylation/dephosphorylation. Its role seems to be an essential control level in addition to the protein phosphorylation by proline-directed kinases. Its cellular function includes regulation of the cell cycle by interaction with phosphorylated mitotic proteins such as Cdc25 and transcription factors such as p53. PIN1 was shown to be involved in the malignant transformation of cells in breast cancer, by up regulation of cyclinD1 and is thought to be involved in the development of the AD by regulating the function of phosphorylated Tau. We propose here to discuss the molecular function of PIN1 at the atomic level based on data from the recent literature and our own results obtained by the technique of Nuclear Magnetic Resonance. PIN1 specifically interacts with pThr/pSer-Pro motifs and is constituted by two domains: a WW N-terminal domain that binds pThr/pSer-Pro epitopes and a prolyl cis/trans isomerase C-terminal catalytic domain. An exception to this organisation is found in the plant PIN1 homologous enzymes, like PIN1At from Arabidopsis thaliana, that are constituted of the sole catalytic domain. The molecular function of PIN1, binding to and isomerization of pThr/pSer-Pro bonds, are thought to lead to several functional consequences. In a first mode of action, exemplified by its competition with the CKS protein, the interaction with PIN1 prevents interaction with other regulatory proteins, like ubiquitin-ligases that lead to degradation pathways. In a second mode of action, the idea is largely accepted that the local isomerization modifies the global conformation of the protein substrate and hence its intrinsic activity, although this has never been directly demonstrated. Finally, isomerization catalysis is thought to regulate the (de)phosphorylation of specific pThr/pSer-Pro motifs, exemplified by the stimulation of the dephosphorylation of pThr231 of Tau by the PP2A phosphatase.

A combined strategy to obtain a partial NMR assignment of the neuronal Tau protein is presented. Confronted with the extreme spectral degeneracy that the spectrum of this 441 amino acid long unstructured protein presents, we have introduced a graphical procedure based on residue type-specific product planes. Combining this strategy with the search for pairwise motifs, and combining the spectra of different Tau isoforms and even of peptides derived from the native sequence, we arrive at a partial assignment that is sufficient to map the interactions of Tau with its molecular partners. The obtained assignments equally confirm the absence of regular secondary structure in the isolated protein.

The interaction between the neuronal Tau protein and the Pin1 prolyl cis/trans-isomerase is dependent on the phosphorylation state of the former. The interaction site was mapped to the unique phospho-Thr231-Pro232 motif, despite the presence of many other Thr/Ser-Pro phosphorylation sites in Tau and structural evidence that the interaction site does not significantly extend beyond those very two residues. We demonstrate here by NMR and fluorescence mapping that the Alzheimer's disease specific epitope centered around the phospho-Thr212-Pro213 motif is also an interaction site, and that the sole phospho-Thr-Pro motif is already sufficient for interaction. Because a detectable fraction of the Pro213 amide bond in the peptide centered around the phospho-Thr212-Pro213 motif is in the cis conformation, catalysis of the isomerization by the catalytic domain of Pin1 could be investigated via NMR spectroscopy.